Fine-mapping A Major QTL Resistance To Gray Leaf Spot In Maize,Gene Prediction And Transcriptome Analysis

Gray leaf spot(GLS)is one of the most destructive foliar maize diseases worldwide,causing substantial yield loss and grain quality degradation.In the last two decades,the disease is quickly becoming prevalent in China,especially in Southwest maize production region,where the GLS is ranked as the most serious maize disease.Generally,the GLS causes 10-30%yield loss,and can reach to 60-80%or even complete yield loss when outbreak.The deployment of GLS-resistant maize hybrids is the most cost-effective and efficient way to control the disease.In our previous study,through using the F2 and F2:3 populations derived from a highly susceptible inbred line Q11 and highly resistant inbred line Y32,a major GLS resistance QTL-qRgls2 was detected in bin 5.03-5.04,with a physical distance of～110 Mb.qRgls2 spans the whole centromere of chromosome 5 and could explain 18.9-23.9%of the total phenotypic variation.In the current study,we used the flanking markers to screen the recombinants within the QTL interval,which were then backcrossed to Q11 to produce backcross progenies.Investigation of both the genotype and phenotype for each individual in the backcross progeny enables us to fine map qRgls2.We conducted this fine-mapping process for several generations to narrow down qRgls2.Due to the low recombination frequency of qRgls2 region,which located near the centromere region,it is very difficult to screen sufficient key recombinants to narrow down qRgls2 into a single candidate gene.To avoid this dilemma,we adopted the association mapping and transcriptome sequencing to confirm the candidate gene underlying qRgls2.The main results are as follows:1.Out of the newly-developed markers,23 were selected to saturate the qRgls2 region for fine-mapping.2.During the years from 2010 to 2011,22 recombinants were screened from the F4 population,which were backcrossed to Q11 to generate 22 backcross progeny,consisting of totally 1,688 BC1F4 individuals.All BC1F4 plants were scored for their GLS disease severities in Baoshan,Yunnan province,and genotyped at the donor regions.Based on both phenotypes and genotypes,the qRgls2 locus was narrowed down to an interval between the markers B96 and x157 with a physical distance of-62 Mb according to B73 RefGen_v2.3.During the period from 2011 to 2012,similar analysis was conducted in the BC1F5-derived BC2F5 progenies.Sixteen markers were used to screen the genotype of 32 BC1F5 recombinants and group them into 18 types.Together with their resistance performance,we restricted qRgls2 to an interval of～3.9 Mb between markers G386 and IDP41.4.During the period from 2012 to 2013,a total of 5,631 BC3F6 plants derived from 42 BC2F6 recombinants were investigated.A total of 14 markers were used to screen the genotype of 42 BC2F6 recombinants and to group them into 17 types.qRgls2 were mapped into the interval between the markers G346 and DD11 with the physical distance of～1 Mb according to the B73 RefGen_v2.5.During the fine-mapping process,the genetic effect of qRgls2 was estimated in 2010-2013.qRgls2 could enhance GLS resistance by 20.6 to 24.6%,and this genetic effect was very stable in multiple populations across different years.6.The genomic sequence between G346 and DD11 was retrieved from B73 RefGen_v2.This region is predicted to contain 15 functional genes according to the 5b.60 annotation of the maize B73 genome v2.Of the 15 predicted genes,one gene GRMZM2G157068 was predicted to encode a calcium-dependent kinase(CDPK).The CDPK family has long been recognized as an essential mediator in diverse abiotic stress as well as innate immunity in maize and other plants.7.Association mapping showed that those gene-derived SNPs significantly associated with GLS resistance did not fall into any of the QTL regions detected in our linkage mapping population.Likewise,the SNPs markers in and around qRgls2 didn’t show significant association with GLS resistance.8.Compared with the control(non-inoculation)samples,multiple differential expressed genes(DEGs)were identified after pathogen inoculationin the two parental lines,Y32 and Q11.Gene Ontology(GO)analysis showed the DEGs were enriched in redox reaction,hormonal regulation and secondary metabolism,suggesting these pathways may play important roles in GLS resistance.Seven out of fifteen functional genes in the qRgls2 region changed their transcripts after inoculation.Taken together,we infer the CDPK gene GRMZM2G157068 is likely to be the candidate for qRgls2.